Mechanism of Jiawei Zhengqi Powder in the Treatment of Ulcerative Colitis Based on Network Pharmacology and Molecular Docking

Objective Ulcerative colitis is an intestinal condition that severely affects the life quality of a patient. Jiawei Zhengqi powder (JWZQS) has some therapeutic benefits for ulcerative colitis. The current study investigated the therapeutic mechanism of JWZQS for ulcerative colitis using a network pharmacology analytical approach. Methods In this study, network pharmacology was used to investigate the potential mechanism of JWZQS in treating ulcerative colitis. The common targets between the two were identified, and a network map was created with the Cytoscape software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of JWZQS was performed using the Metascape database. Protein-protein interaction networks (PPI) was created to screen core targets and main components, and molecular docking was conducted between the main components and core targets. The expression levels of IL-1β, IL-6, and TNF-α were detected in animal experiments. Their effect on the NF-κB signaling pathway and the protective mechanism of JWZQS on the colon by tight junction protein were investigated. Results There were 2127 potential ulcerative colitis targets and 35 components identified, including 201 non-reproducible targets and 123 targets shared by drugs and diseases. Following the analysis, we discovered 13 significant active components and 10 core targets. The first 5 active ingredients and their corresponding targets were molecularly docked, and the results showed a high level of affinity. GO analysis showed that JWZQS participate in multiple biological processes to treat UC. KEGG analysis showed that JWZQS may be involved in regulating multiple pathways, and the NF-κB signaling pathway was selected for analysis and verification. JWZQS has been shown in animal studies to effectively inhibit the NF-κB pathway; reduce the expression of IL-1β, TNF-α, and IL-6 in colon tissue; and increase the expression of ZO-1, Occludin, and Claudin-1. Conclusion The network pharmacological study provides preliminary evidence that JWZQS can treat UC through multiple components and targets. JWZQS has been shown in animal studies to effectively reduce the expression levels of IL-1β, TNF-α, and IL-6, inhibit the phosphorylation of the NF-κB pathway, and alleviate colon injury. JWZQS can be used in clinical, but the precise mechanism of UC treatment requires further investigation.


Introduction
Ulcerative colitis (UC) is an inflammatory condition that primarily affects the mucosa and submucosa of the large intestine (rectum and colon). It is distinguished by large intestinal mucosal ulceration and persistent inflammation [1]. Mucous, diarrhea, hemafecia, pus, stomachache, and tenesmus were the most common clinical symptoms [2]. Medication, surgery, and antibody therapy are the three main types of current UC treatments. The most common existing therapies include aminosalicylates, corticosteroids, and immunosuppression, all of which have negative side effects and necessitate long-term medication [3]. Therefore, safer and more effective therapeutic approaches for UC are required.
In traditional Chinese medicine (TCM), there are several ways to treat UC, which is also known as diarrhea or dysentery. TCM provides a comprehensive understanding of a patient's physical condition based on their pulsation, tongue, signs and symptoms, and modern laboratory tests. Although therapeutic regimens vary depending on constitution, the general approach is to clear heat; expel dampness; strengthen the spleen, kidney, and qi; promote blood circulation; and dispel blood stagnation. JWZQS decoction contains Houpo, Chenpi, Banxia, Danggui, Chuanxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma. The original prescription comes from a Ming Dynasty work by Xue Ji called the "Waike Jingyao." It has Qi-widening, spleen-energizing, and dampening effects. The current study makes reference to Andrew L. Hopkins' theory of "network pharmacology" and Professor Li Shao's theory of "correlation between TCM and biomolecular network" [4,5]. Network pharmacology, which combines pharmacology and systems biology, is an analytical system suitable for the study of complex components of traditional Chinese medicine.
The current study used the network pharmacology approach to construct the "drug-components-targets" network. For the core target and pathway verification, the KEGG and GO enrichment analyses were performed. Molecular docking technique was used to study the affinity between active components and key targets in JWZQS. The flow chart for the study is shown in Figure 1.
We used dextran sulfate sodium salt (DSS) to cause colon injury in mice, developed a UC mouse model, and assessed the clinical symptoms of the mice. Mouse colons were collected, colonic lengths were measured, and the contents and expressions of TNF-α, IL-6, and IL-1β in tissues were measured. The phosphorylation of IκB-α and P65 in the NF-κB pathway was detected. Furthermore, the tissue was sliced to examine the damage to the colon. Immunohistochemistry (IHC) detected the expression of ZO-1, Occludin, and Claudin-1 in the mouse colon tissue.

Article Types
The article type was original research.

Collection of Components and Targets of JWZQS.
We use the search terms " Houpo, Chenpi, Banxia, Danggui, Chuanxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma" in the TCM database of system pharmacology (TCMSP, http:// tcmspw.com). Following the bioactive constituents criteria, active constituents with drug similarity (DL) not less than 0.18 and oral bioavailability (OB) not less than 30% were screened [6]. Based on TCMSP, we identified potential targets of active JWZQS constituents. Using the UniProt database, species were restricted to "Homo sapiens," and annotation normalization was applied to the target genes [7].

Construction of Drug-Components-Targets Network.
Cytoscape software (3.10.1 version) is used to associate drugs, active ingredients, targets, and diseases and to build an interaction network diagram between components and targets. Edges represent node relationships, while nodes represent active ingredients and targets. The main active components of the drugs were then examined.

Construction of PPI Network and Screening of Core
Targets. To establish the interaction between the two proteins, the common target was imported into the Cytoscape database (https://cytoscape.org), and restriction conditions were set for the human species [13]. The database calculated the interrelationship, closeness, degree, feature vector, average local connectivity, and network for network analysis. Finally, the traditional "degree" value was employed to assess the importance of PPI network nodes.

Analysis of Enrichment and Associated Signaling
Pathways. To study JWZQS's biological process and signaling pathway in managing UC, we used the Metascape website to analyze molecular function (MF), biological process (BP), and cell component (CC) data. Their sequencing is determined by the number of targets involved in biological processes and signaling pathways.
AutoDock Vina 1.1.2 was used in the current study to perform molecular docking [15]. PyMol 2.5.4 was used to remove small molecules, salt ions, and water molecules from the receptor proteins before docking [16]. A docking box was created to contain the entire protein architecture. The entire processed receptor proteins and small molecules were converted into the PDBQT format via ADFR suite 1.0 to prepare AutoDock Vina 1.1.2 docking [17]. While connecting, keep the default settings for interconnection parameters. The binding confirmation was determined to be the output docking confirmation with the highest score. The final step involved a visual analysis based on PyMol 2.5.4 docking results.         The SASP (Shanghai Xinyi Co. LTD.) group 100 mg/kg SASP suspension by gavage [19]. Gavage was used to administer the corresponding volume of distilled water to the NT group.

Clinical
Scoring and Sample Collection. The disease activity index (DAI) scoring system (Table 1) was used to determine body weight (BW), fecal occult blood, fecal char-acteristics, and clinical symptoms. Finally, colons of mice were collected for relevant experimental analysis [20]. 3.10. qRT-PCR Assay. Colon tissues from mice were collected and treated to extract mRNA, and the levels of IL-1β, TNF-α, and IL-6 were determined using qRT-PCR as previously described [21]. Colon tissues from mice were collected and treated to extract mRNA, and the levels of IL-1β, TNF-α, and IL-6 were determined using qRT-PCR as (c) Figure 2: (a) Venn diagram representing the potential targets for JWZQS against UC. (b) "drug-components-targets" network. The orange represents the drug; the surrounding balls represent its components; A1, B1, and C1 represent the common components between drugs; and the diamond represents genes. (c) "Drug-components-targets-disease" network. The blue represents UC, the orange represents the disease target, the green represents JWZQS, and the pink represents the active components. 6 BioMed Research International 7 BioMed Research International previously described [21]. Table 2 lists all of the substrates that were used. The arithmetic formula 2 −ΔΔCT was used to obtain the relative quantitative results.
3.12. Hematoxylin and Eosin (H&E) Staining. Tissues from mouse colons were fixed in 4% formaldehyde and paraffin before being sliced into 5 μm sections for H&E staining. After staining, the histology was examined under a light microscope. Finally, histological scores were computed following Table 3 [22].
3.13. Immunohistochemistry (IHC). Immunohistochemical analysis of mouse colon tissue was performed to observe the contents of ZO-1, Occludin, and Claudin-1 proteins in mouse colon tissue to further understand the protective mechanism of JWZQS on mouse colon. The sections were incubated in a citrate antigen repair solution for 20 min at 95°C. These sections were incubated overnight with the primary antibodies ZO-1 (D6L1E 1 : 100), Occludin (ab216327 1 : 100), and Claudin-1 (ab15098 1 : 100) and then with the secondary antibodies for 50 min before being examined under a microscope.
3.14. Statistical Analysis. The data is presented as the mean ± SD. Prism 8.0 was used to compare two groups using unpaired Student's t-tests. Prism 8.0.20 was used to perform ANOVA (general linear model) comparisons of more than two groups. Nonparametric tests must be used to evaluate clinical and pathology scores.

Collection of Active Components of JWZQS and Study of
Overlapping Targets. The following nine Chinese medicines were searched in the TCMSP database: Houpo, Chenpi, Banxia, Danggui, Chuangxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma. These compounds' screening criteria were DL (≥0.18) and OB (≥30%). The TCMSP database was used to screen the targets linked to active ingredients, after which the target information was compared, and the gene name was adjusted using the UniProt database [23]. After matching, 670 targets were discovered, and 201 targets were discovered after removing duplicate values, as shown in Table 4.
Using the search term "ulcerative colitis," the human genome annotation databases GeneCards, OMIM, DisGe-Net, and DrugBank were searched for disease-related genes.

BioMed Research International
There were 2127 acquired disease targets. The acquired target genes were compared with the genes associated with the aforementioned drug active ingredients. The String website generated a Venn diagram to screen for common genes, as shown in Figure 2(a).

Construction of "Drug-Components-Targets," "Drug-
Components-Targets-Disease," and "PPI" Network Maps and Screening of Core Targets. The obtained active ingredient and genetic data will be used to generate "Drug-Components-Targets" and "Drug-Components-Targets-Disease" network maps using Cytoscape software. Figures 2(b) and 2(c) depict the intuitive relationship between drugs, diseases, and targets. Thirteen components, including quercetin, naringenin, wogonin, nobiletin, and irisolidone, were linked to disease targets and may be useful in the treatment of UC.
With the common gene data imported, the String database was used to generate a protein-protein interaction network (PPI) interaction map with the species "Homo Sapiens" as the designation. The TSV file was exported with the lowest interaction score (0.7). The Cytoscape software was used to calculate the major protein interactions. We imported the 123 common targets in the intersection into BCL2L1 CD4 0LG BI RC 5 EG FR B A X M A P K 1 4  10 BioMed Research International the String database to generate the interaction map. We import the interaction diagram into Cytoscape software to generate the PPI network diagram (Figure 3(a)).

GO and KEGG Analyses.
The GO function and KEGG pathway of JWZQS were investigated using Metascape to better understand the compound's treatment mechanism on UC. In terms of BP, the therapeutic effect of JWZQS on UC is mainly due to cellular responses to lipid, hormones, substances, lipid peptides, radiation, positive regulation of cell motility, and other factors. Protein homodimerization activity, cytokine activity, transcription factor binding, protein domain-specific binding, DNA binding, kinase binding, and other activities are all part of the MF project. In terms of CC, JQZQS may influence membrane rafts, transcription regulator complexes, membrane sides, vesicle lumens, and other cell structures. JWZQS may be used to treat UC by interfering with these BP (Figure 4(a)). Cancer, lipid and atherosclerosis, diabetic complications, chemogenic receptor activation, cellular senescence, platinum resistance, and diabetic cardiomyopathy were the most affected pathways in the KEGG analysis (Figure 4(b)). We discovered numerous associations between rich pathways and additional pathological effects, which could be attributed to shared molecular targets in various diseases (Figure 4(c)). JWZQS may regulate the expression of TNF-α, IL-6, and IL-1β in the NF-κB pathway. It may control the phosphorylation of IκB-α and P65 to prevent inflammation ( Figure 5).

Molecular Docking.
We discovered in the PPI network diagram that the core targets were AKT1, IL6, TP53, TNF, and IL-1β. In the "drug-components-targets-disease" network diagram, quercetin, naringenin, wogonin, nobiletin, and irisolidone are discovered to be the main active components of JWZQS. We used molecular docking to find their corresponding relationship in the drug-components-  Figure 2(c), Vina 1.1.2 was used to investigate the docking of compounds irisolidone, naringenin, nobiletin, quercetin, and wogonin with IL-1β, AKT1, TNF, IL-6, and TP53 proteins, respectively ( Figure 6).
TA negative binding affinity indicates the likelihood of binding, and when the affinity value is less than -6 kcal/ mol, the binding is frequently thought to be highly likely. Docking scores for AKT1 and naringenin are -8.1 (kcal/ mol), IL-1β and irisolidone are -6.4 (kcal/mol), IL-6 and

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BioMed Research International quercetin are -7.4 (kcal/mol), TNF and nobiletin are -9.1 (kcal/mol), and TP53 and wogonin are -6.6 (kcal/mol). It is worth noting that AKT1 has a high affinity with naringenin and TNF with nobiletin. Then, we dock all ligands for each docked protein, to observe the interaction between the core targets and other active components (Table 5).

JWZQS Inhibited the Expression of the NF-κB Pathway.
We examined the levels of inflammatory factors in the colons of mice in each group. We discovered that JWZQS could inhibit the expression of inflammatory factors, such as IL-6, IL-1β, and TNF-α in the MOD group, effectively controlling inflammation development (Figure 7). Protein bands from the NF-κB pathway were analyzed to investigate the protective mechanism of JWZQS in UC mice (Figure 8(b)). JWZQS effectively inhibited IκB-α and P65 phosphorylation in the NF-κB pathway (Figure 8(a)). Therefore, JWZQS can inhibit the NF-κB signaling pathway activation and reduce colon inflammation in mice.

JWZQS Showed a Good Protective Effect on DSS-Induced UC Mouse
Model. Images of colon samples revealed that fecal urine appeared in the colons of MOD group mice, and the colon length was significantly reduced (Figures 9(a) and 9(b)). Every day, the clinical symptoms of mice were recorded (Figure 9(c)). Every day, except for the blank group, the weight of the mice was recorded (Figure 9(d)). The MOD group's average colon length was shorter than that of the other groups. H&E staining was used to stain mouse colon specimens. After staining, the histology was examined under a light microscope, and histological evaluations were carried out. The MOD group mice had fewer colonic goblet cells, lost colonic crypts, and typical edematous infiltration of inflammatory tissue when compared to the other groups ( Figure 10(a)). The results revealed that the content of MOD histone decreased significantly, and the SASP group clearly outperformed the MOD group, as did the Chinese medicine dose groups, which also outperformed the MOD group and demonstrated a concentration gradient advantage (Figure 10(b)).

Discussion
UC is currently classified as a refractory condition by the medical community. Mucous pus, diarrhea, hemafecia, and stomachache are some of the clinical symptoms. Its lesions mostly infiltrate the rectal and colonic mucosa/submucosa. Clinical characteristics include a long disease course, ease of recurrence, a difficult prognosis, and numerous complications [24]. Despite its low prevalence, UC is on the rise, and it is a refractory disease with a lengthy treatment cycle and a high fatality and disability rate [25]. The current oversimplified Western drug therapies are not only costly but also heavily dependent and drug-resistant. Therefore, it is critical to improve treatment methods and encourage the development of new, more effective therapeutic options with fewer side effects.
Along with the growth of network pharmacology, a new method for locating therapeutic agents has recently emerged. The multitarget activity in network pharmacology corresponds to the complex disease pathways and drug action. Understanding TCM through network pharmacology is a growing trend [26]. Network pharmacology is a cutting-edge research method that combines network science, bioinformatics, and systems biology to assess molecular associations between pharmaceuticals and therapeutic entities at the physiological network and system level facets and clarify drug methodological pharmacodynamics. Exploring such intricate TCM constituents is a natural fit for its multichannel and multitarget properties [27]. Therefore, the current study established the mechanism of action of JWZQS in methodologically managing UC.
The analysis of "drug-components-targets" and "drugcomponents-targets-disease" revealed 13 active ingredients, which are key ingredients in the treatment of UC. The PPI network discovered that AKT1, IL6, TP53, TNF, and IL-1β are core targets, and their interactions are critical to the completion of various biological processes. GO and KEGG analyses revealed that JWZQS's anti-UC effect is primarily derived from the cellular response to positive regulation of lipids, hormones, substances, lipid peptides, radiation, and cell movement. Some examples include protein homodimer activity, cytokine activity, transcription factor binding, protein domain-specific binding, DNA binding, kinase binding, and other MF items. JQZQS may influence membrane rafts, transcriptional regulatory complexes, membrane sides, vesicle lumens, and other cellular structures in CC. JWZQS may be used to treat UC by interfering with these biological processes. JWZQS are primarily involved in regulating cancer, and IL-1β may be regulated in the NF-κB pathway. It also influences IκB-α and P65 phosphorylation. Animal studies will confirm this later. Irisolidone, naringenin, nobiletin,